Generation of cellular asymmetry is required for transmission of nerve impulses, embryonic development, transport of molecules across an epithelial layer, cell migration in normal and metastatic cells, and cell division. The cytoskeleton plays an essential role in these processes as a force generator for intracellular and cellular movement, a scaffold to stabilize asymmetric cell structure, and a track for directed intracellular movement. An important clue to understanding cytoskeletal organization during establishment of cell polarity came from our studies on actin dynamics in living yeast cells. We find that actin patches and cables, the major components of the actin cytoskeleton in yeast, achieve polarization by assembly at sites of polarized cell surface growth, and in the case of actin cables, extension along the mother-bud axis. Moreover, we obtained evidence for a role of myosin I proteins in this process. Myosin I proteins were discovered over 25 years ago. Nonetheless, the functions of many of these proteins are unknown, and the limited evidence for myosin function and roles provides no information on their mechanism of action. In the budding yeast, there are two functionally redundant myosin I genes, MYO3 and MYO5. Deletion of both genes produces disorganization of actin cables and actin patches, and defects in actin-dependent processes including secretion, endocytosis, and polarized cell growth and division. Consistent with this, we and others find that 1) myosin I proteins localize to sites 0 polarized cell surface growth, 2) verprolin and Las17p/Bee1p, a protein that activates the actin nucleation activity of the Arp2/3 complex, bind to myosin I proteins and contribute to targeting of myosin I proteins to sites of polarized cell surface growth, and 3) myosin I proteins can bind to and activate the Arp2/3 complex. Future studies on the mechanism of my osin I-mediated actin assembly will focus on the relation between actin cables and patches, the mechanism underlying actin cable and patch assembly and disassembly, and the role for myosin I proteins and other cytoskeletal organizers in assembly, disassembly and dynamics of the actin cytoskeleton. Given the fundamental role of the actin cytoskeleton in establishment of cell polarity, and evidence for a role of myosin I proteins in actin nucleation and actin-based movement, it is surprising that we know so little regarding the function of myosins and their precise role in actin cytoskeletal organization. The studies proposed are designed to fill that gap.